The global health burden of annual influenza epidemics coupled with emerging influenza pandemics highlight the urgent need for new, effective treatments. The majority of current circulating seasonal influenza viruses, as well as highly pathogenic avian influenza viruses such as H5N1 and H7N9, carry the S31N mutant in their M2 genes, which confers resistance to adamantanes (amantadine and rimantadine). Influenza viruses that are resistant to oseltamivir, the only orally available drug, are continuousy on the rise. Thus there is a persistent need for novel anti-influenza drugs. The M2-S31N mutation is persistent in more than 95% of currently circulating influenza A viruses, and it is one of the most conserved viral proteins. Therefore inhibitors targeting M2-S31N will be excellent candidates for the next generation of anti-influenza drugs. This proposal focuses on developing M2-S31N channel blockers with favorable drug-like properties and in vivo activities. We implement a multi-faceted approach that includes structure-based drug design, medicinal chemistry, electrophysiology, and virology to design M2-S31N channel blockers. The goal is to develop broad spectrum antiviral drugs that are active against clinical isolates of influenza A viruses that are resistant to one or both classes of currently approved anti-influenza drugs. In the R21 phase we will focus on optimizing our previously discovered M2-S31N inhibitors as well as exploring novel chemotypes. The inhibitor design will be guided by our previously solved solution NMR structure of M2-S31N. Synthesized molecules will be tested in antiviral assays against clinically relevant influenza A viruses that show resistance to either amantadine or oseltamivir or both. The mechanism of action will be confirmed using electrophysiological assays and solution NMR NOESY spectroscopy. To prioritize compounds for the in vivo studies, we will profile the ADME properties of top lead compounds as well as evaluate their tendencies to elicit drug resistance. In the R33 phase, we will evaluate the combination therapy potential of M2-S31N inhibitors with oseltamivir or ribavirin. The structure-activity relationship studies will e driven by the in vitro ADME properties, cytotoxicity, and synergistic effects with oseltamivir or ribavirin. We will assay the in vivo pharmacokinetic ADME properties of up to five compounds in mice. For efficacy studies, mice will be challenged with a lethal dose of mouse-adapted influenza A viruses, and M2-S31N inhibitors will be administrated by oral gavage or tail vein injection. Survival rate, body weight loss, and other clinical signs will be used to evaluate the i vivo efficacy of M2-S31N inhibitors. Upon successful completion of the proposed studies, we will have at least one compound that is ready for the next stage of studies needed for Investigational New Drug (IND) filling.